分子水平上解析落叶松枯梢病扩散蔓延规律

[Objective]Clarifying the diffusion and spread patterns of larch wilt disease,as well as the genetic differentiation of its pathogenic pathogen,Neofusicoccum laricinum,during the transmission process,is of great significance for the prevention and control of larch wilt disease.[Method]The surface disinfection method was used to isolate the pathogenic fungal at the disease-health junction of samples of Larch wilt disease from multiple provinces and diverse regions across the country,and combined with morphology and molecular biology,they were identified.Koch's rule verifies the pathogenicity of representative strains and observes the growth of N.laricinum under different conditions,such as culture medium,carbon source,nitrogen source,pH,and light,to analyze its biological characteristics.Using third-generation sequencing technology to sequence the genomes of representative strains and resequencing the genomes of other geographical races based on multi-genome SNP data,analyze the genetic diversity and geographic information of Larix gmelinii.Determine SNP-high polymorphism target DNA fragments and DNA barcodes suitable for the identification of geographical races of Larix gmelinii.Finally,systematically and comprehensively analyze the transmission law and genetic characteristics of Larix gmelinii wilt disease.[Result]From 2022 to 2023,a total of 101 tree farm samples from 7 provinces,including Liaoning,Jilin,Heilongjiang,Inner Mongolia,Hebei,Gansu,and Shandong,were collected.Combining morphology and molecular biology,62 strains of N.laricinum have been isolated and identified from 17 forest farms in Heilongjiang Province,Jilin Province,and Inner Mongolia Autonomous Region,with 7 forest farms in Heilongjiang Province and Jilin Province each.Two fungal strains,HLJ-QQQHE-4 from Qiqihar,Heilongjiang,and JL-TM-2 from Tumen,Jilin,were selected for pathogenicity testing on larch twigs.After 3 days of inoculation,there was a phenomenon of chlorosis at the inoculation site.After 15 days,the twigs completely withered and drooped,further confirming that the isolated pathogen was the pathogen of larch shoot blight.Research on biological characteristics has found that under alkaline conditions,with the addition of 30 g/L soluble starch and 2 g/L ammonium sulfate and continuous illumination,the growth state of N.laricinum is optimal.The Hi-C assembly results showed that the genome of the HLJ-QQQHE-4 strain from Aihua Forest Farm in Kedong County,Heilongjiang Province,has 14 chromosomes,a GC content of 53.34%,a genome size of 42.47 Mb,and only one small gap.The sequencing quality is high,with a total of 11275 genes encoding the protein.Evolutionary analysis shows that this strain has the closest evolutionary relationship with the same monophyletic group as N.parnum,further proving its taxonomic status.[Conclusion]The identification and pathogen isolation techniques for this disease have matured and been mastered.Analysis has found that Jilin and Heilongjiang have a large incidence area.Genome sequencing provides a reference for the study of the genetic characteristics of this fungal.The optimal growth conditions selected also lay the foundation for the later cultivation and research of this fungal.To complete the molecular-level analysis of the diffusion and spread of larch wilt disease,subsequent experiments are still ongoing.